S. aureus USA300 is a formidable human pathogen isolated from the majority of human skin and soft tissue infections in the US. We postulate that the keratinocyte is responsible for much of the pathology induced by USA300 infection and is a potential target for prevention or therapy. In Aim #1 we will establish that the expression of a specific group of staphylococcal gene products by USA300 and the sequenced clinical isolates that we will study facilitates the endocytosis of the organisms by human keratinocytes, their ability to escape from the endosome and to activate caspase-1 mediated inflammasome signaling. Staphylococcal induction of keratinocyte pyroptosis, release of IL-1 and HMGB1 stimulate the local accumulation of neutrophils causing the characteristic pyogenic infection. We expect that blocking keratinocyte pyroptosis pharmacologically could prevent S. aureus invasion through the keratinocyte barrier. Staphylococcal endocytosis is mediated primarily by ?51 integrins that activate PI3K-Akt signaling and proliferation, indicating the likelihood that S. aureus infection also triggers keratinocyte proliferation. In Aim #2 we will determine how actively replicating keratinocytes with increased availability of ?51 surface receptors contribute to the bacterial uptake, thus fueling persistent local infection. In the third aim, using human keratinocytes in organotypic cultures in vitro and in SCID:hu mice with human skin xenografts, we will establish if targeting keratinocyte signaling; blocking keratinocyte pytoptosis with caspase-1 or calpain inhibitors and blocking PI3K-Akt mediated keratinocyte proliferation will prevent pyroptosis and subsequent neutrophil recruitment. These studies would provide novel targets to prevent or treat S. aureus skin infection. Public Health Implications: Staphylococcal skin infections are among the most common reasons for ER visits, associated with tremendous morbidity and cost. Although even the USA 300 MRSA is still susceptible to many antibiotics, these organisms nonetheless persist. This project will determine if keratinocyte signaling, which we postulate is responsible for much of the pathology evoked by these organisms, provides therapeutic targets to prevent staphylococcal infection.